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Investigation of abnormal haemoglobins and thalassaemia

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... Many variants of haemoglobins have no critical haematological and clinical significance because the underlining mutation causes no alteration in the function, solubility, or stability of the haemoglobin molecule [1]. Some of these mutations however affect the physical or chemical properties of the haemoglobin molecule resulting in haemoglobin solubility, instability, or oxygen-binding properties. ...
... Some of these mutations however affect the physical or chemical properties of the haemoglobin molecule resulting in haemoglobin solubility, instability, or oxygen-binding properties. Haemoglobin variants such as Hb-S, Hb-C, Hb-D Punjab , Hb-E, and Hb-O Arab have clinical or genetic significance and are readily detectable by electrophoretic and chromatographic techniques [1]. ...
... Most diagnostic laboratories do not differentiate them. Hb-C is isopropanol stable while Hb-E is slightly unstable forming precipitate in isopropanol [1] [3]. This study investigated the percentage of haemoglobin instability in Hb-AC, Hb-SC, and Hb-CC individuals and the possibility of Hb-E individual erroneously phenotyped as Hb-C. ...
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One of the limitations in haemoglobin electrophoresis technique is the inability to distinguish between stable haemoglobin-C and unstable Haemoglobin-E.Haemoglobins A2, C, E and OArab migrate together duringcellulose acetate electrophoresis and are often not differentiated by most diagnostic laboratories. This study investigated the instability of phenotyped haemoglobin-C individuals and the possibility of haemoglobin E individual phenotyped as either heterozygous or homozygous haemoglobin-C. Fifty phenotyped blood samples consisting of twenty five haemoglobin- AC, fifteen haemoglobin -SC, and ten haemoglobin - CC were used for this study. Twenty two blood samples phenotyped as haemoglobin – AA served as controls. The blood samples were analyzed For haemoglobin instability using heat instability and isopropanol instability tests. The difference in turbidity of the haemoglobins measured spectrophotometrically before and after heating was taken as a measure of haemoglobin instability. The degree of instability in haemoglobin-AC individuals was insignificant (p>0.05) in relation the controls . A significant (p<0.05) level of instability was found in haemoglobins - SC and CC individuals. Two haemoglobin-AC and one haemoglobin - CC individuals showed very high concentration of haemoglobin instability. They recorded turbid reaction with isopropanol. This suggests Haemoglobin -S,E,Haemoglobin CE or Haemoglobin-EE erroneously phenotyped as haemoglobin -SC and haemoglobin-CC. This study concludes that Haemoglobin-E may be present in Nigeria.
... 1,3,4 Sindroma ini menyebabkan gangguan klinis yang jauh lebih berat dibandingkan dengan HbE homozigot tetapi lebih menyerupai thalassemia b mayor. [5][6][7] Ada beberapa metode untuk investigasi sangkaan thalassemia dan Hb varian tetapi diagnosa umumnya sudah dapat ditegakkan berdasarkan gambaran hematologi dan analisa hemoglobin. 4,6,8,9 Elektroforesis hemoglobin pada pH alkali merupakan salah satu cara pemeriksaan analisis hemoglobin yang banyak digunakan untuk membuktikan adanya hemoglobin abnormal. ...
... [5][6][7] Ada beberapa metode untuk investigasi sangkaan thalassemia dan Hb varian tetapi diagnosa umumnya sudah dapat ditegakkan berdasarkan gambaran hematologi dan analisa hemoglobin. 4,6,8,9 Elektroforesis hemoglobin pada pH alkali merupakan salah satu cara pemeriksaan analisis hemoglobin yang banyak digunakan untuk membuktikan adanya hemoglobin abnormal. Kadar Kadar HbA 2 yang meningkat 3,6-7,8% pada seorang yang secara klinis normal, merupakan kriteria diagnostik thalassemia b trait. ...
... Di kasus HbE heterozigot yang khas kadar HbA 2 berkisar 20-35% tempat hemoglobin E pada elektroforesis biasanya bergerak dan bertumpu pada HbA 2 . 4, [6][7][8] Angka kejadian thalassemia dan hemoglobinopati dapat diperkecil dengan cara konseling genetik dan diagnosis prenatal. �leh karena itulah kajian keluarga sangat diperlukan dalam rangka penyaringan genetik untuk mencegah thalassemia dan hemoglobinopati. ...
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HbE-b thalassemia is an inherited hemoglobin disorder of gene combination b thalassemia and HbE. It is caused by thalassemia and hemoglobinopathy gene,which acting as the allele in the same locus of chromosome. The clinical presentation is more severe than HbEhomozygote and almost similar to b thalassemia major. This disease is found predominantly in South East Asia, including Indonesia.The family study is very needed in order to genetic screening to prevent hemoglobinopathy and thalassemia homozygote. The objective ofthis study is to know the pedigree from the daughter who suffer from HbE- b thalassemia which is taken care in the children ward at H.Adam Malik Top Refferal Hospital, Medan. The family study was also done to both father and mother, one sister and to both grandparent.An examination was carried ,which include physical examination, complete cell blood count, peripheral blood morphology, hemoglobinanalysis with Hb-electroforese pH 8.5 agarose medium and read with densitometri, and osmotic fragility examination. From the familystudy was found that her father suffer from HbE heterozygote while the mother suffer from b thalassemia trait and sister got HbE- bthalassemia. The father and mother have no relative acceptance and there is no any blood linkage. Her grandparent could not performthe blood sample because they were have already passed away .
... Children of the b-thal trait individuals can inherit b-TM if their partner also has b-thal trait. Patients with b-TM require a lot of financial, social, and psychological support for the maintenance and treatment of their illness [3,21]. The estimated prevalence of b-thal and sickle cell anemia in KSA is 1.0-5.0% ...
... To reduce the burden of the disease on future generations, KSA initiated the PMS program in the year 2004. However, the initial studies have shown a poor knowledge of the PMS program among the general population as well as specific groups for example unmarried population and university students [2,16,21,27]. The consanguinity rate in KSA is around 56%. ...
Article
The National Premarital Screening Program, which includes sickle cell disease and thalassemia, was made mandatory in 2004 by the Kingdom of Saudi Arabia (KSA), and the earlier studies have shown a poor knowledge and negative attitude toward this program in the different study groups. This study was conducted to assess the knowledge and attitudes toward premarital screening (PMS) in a randomly selected national sample of the Saudi population, 18 years and above. This was a cross-sectional study conducted in the Saudi population in the western region between July and December 2021. Valid and reliable questionnaire and data were collected from 893 participants aged ≥18 years. The χ² test was used to ascertain if there is an association between categorical variables. Multivariate logistic regression was used to determine factors predicting satisfactory knowledge. All 893 study participants had heard about PMS with 625 (70.0%), 244 (27.3%) and 24 (2.7%) having satisfactory, fair and poor knowledge, respectively. Participants aged 26–35 years (p =0 .038), females (p < 0.001), those with higher education (p = 0.003) and employed (p = 0.004), had a better knowledge compared to other groups. Most of the participants had a positive attitude toward PMS. There is a changing trend in the knowledge and attitude toward PMS with a greater number of people wanting to go for PMS. There is also an improvement in the number of participants opting out of marriage in case of incompatibility with their future partner. However, the health education programs need to be improved regarding the hemoglobinopathies.
... The physicochemical principle is based on insolubility and polymerization of deoxy HbS, which forms the pathophysiologic basis for Hb solubility and red cell sickling tests. 8 The electrochemical principle is based on charge-mobility alterations in HbS, which forms the pathophysiologic basis for Hb electrophoresis, isoelectric focusing, and chromatography. 8 SCD arises from the homozygous inheritance of HbS (HbSS) or double heterozygosity with another hemoglobinopathy (e.g., HbSC, HbSD, HbSE, HbSO, and HbSβthal). 1 The clinical course of SCD is characterized by a painless period of relative well-being referred to as 'steady-state' , which is intermittently interrupted by painful periods referred to as 'vaso-occlusive crisis' (VOC). ...
... 8 The electrochemical principle is based on charge-mobility alterations in HbS, which forms the pathophysiologic basis for Hb electrophoresis, isoelectric focusing, and chromatography. 8 SCD arises from the homozygous inheritance of HbS (HbSS) or double heterozygosity with another hemoglobinopathy (e.g., HbSC, HbSD, HbSE, HbSO, and HbSβthal). 1 The clinical course of SCD is characterized by a painless period of relative well-being referred to as 'steady-state' , which is intermittently interrupted by painful periods referred to as 'vaso-occlusive crisis' (VOC). 9 Clinical transition from steady-state to VOC is usually triggered by various factors that range from physiological (e.g., menstruation) to nonphysiological (e.g., infection) factors on the one hand, and from psychological (e.g., mental stress) to physical (e.g., physical exhaustion) factors on the other hand. ...
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Apart from hemoglobin-S (HbS), there are other Hb variants (non-S sickling Hb variants) that cause sickle cell disease. However, the profiles of these non-S sickling Hb variants have neither been collated nor harmonized. A literature search revealed 14 non-S sickling Hb variants (HbC-Harlem, HbC-Ziguinchor, HbS-Travis, HbS-Antilles, HbS-Providence, HbS-Oman, HbS-Cameroon, HbS-South End, Hb Jamaica Plain, HbC-Ndjamena, HbS-Clichy, HbS-San Martin, HbS-Wake, and HbS-São Paulo). Generally, the non-S sickling Hb variants are double mutants with the HbS mutation (GAG>GTG: βGlu6Val) and additional β-chain mutations. Consequently, non-S sickling Hb variants give positive solubility and sickling tests, but they differ from HbS with respect to stability, oxygen affinity, and electro-chromatographic characteristics. Similarities and discrepancies between HbS and non-S sickling Hb variants create diagnostic pitfalls that can only be resolved by elaborate electro-chromatographic and/or genetic tests. It is therefore imperative that tropical hematologists should have a thorough understanding of these atypical sickling Hb variants. Collated and harmonized appraisal of the non-S sickling Hb variants have not been previously undertaken. Hence, this paper aims to provide a comprehensive but concise historical, genetic, comparative, diagnostic, and clinical overview of non-S sickling Hb variants. The elaborate techniques often required for precise diagnosis of non-S sickling Hb variants are regrettably not readily available in low resource tropical countries, which paradoxically carry the heaviest burden of sickling disorders. We strongly recommend that tropical countries should upgrade their diagnostic laboratory facilities to avoid misdiagnosis of these atypical Hb mutants.
... Consequently, insufficient haemoglobin is produced and severe anaemia occurs in the early life of these patients. [2] Beta-thalassemia major, which is clinically the most severe form and the affected children are dependent on regular red blood cells transfusion for survival. [3] Patients with beta-thalassemia major require repeated red blood cells transfusion therapy in order to maintain their haemoglobin level between 10-12 gm/dL and survive. ...
... We know that each unit of conventional packed red blood cells transfusion adds about 200-250 mg of iron extra to the recipients. [1,2,4] As in the study, numbers of neocytes concentrates/pooled neocytes transfused to the study group patients are quite lesser than the numbers of conventional packed red blood cells transfused to the control group patients, so serum iron reduction observed in the neocytes concentrate/pooled neocytes transfusion cases than conventional packed red blood cells transfused cases. The serum iron reduction was observed 5/12 X 100% =41.67% in study group in comparison to control group. ...
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BACKGROUND Beta-thalassemia major patients require continuous red blood cells transfusion therapy for maintaining their haemoglobin level between 10-12 gm/dL and survive. Each unit of red blood cells (About 180 ml) transfusion adds chances of transfusion related hazards and adds about 200-250 mg of extra irons to the recipients. Previous studies of neocytes concentrate/pooled neocytes transfusion demonstrated decreased numbers of required red blood cells transfusion by extending transfusion intervals, decreased incidences of transfusion related hazards and reduction of iron overload in these patients.
... Result for haemoglobin S variant was noted down after 5 minutes. The sample with turbidity was considered positive for sickle cell disorder (Wild and Bain, 2006). Sample from known case of sickle cell trait was used as positive control. ...
... All the samples were subjected to haemoglobin electrophoresis using cellulose acetate membrane in alkaline TEB buffer at pH 8.9 for electrophoresis pattern (Wild and Bain, 2006). Control of known sickle cell trait (AS) patient was used for electrophoresis. ...
... The Hb phenotypes of subjects were determined by haemoglobin electrophoresis at a pH of 8.6 on cellulose acetate paper, sickling test and haemoglobin quantitation (21) . On the basis of the electrophoretic patterns, the patients phenotypes were categorized as normal (Hb AA) or SCT (Hb AS) (21) . ...
... The Hb phenotypes of subjects were determined by haemoglobin electrophoresis at a pH of 8.6 on cellulose acetate paper, sickling test and haemoglobin quantitation (21) . On the basis of the electrophoretic patterns, the patients phenotypes were categorized as normal (Hb AA) or SCT (Hb AS) (21) . The ABO blood groups of patients were determined manually by using monoclonal anti-A and anti-B against the subjects red cells suspended in saline tubes at room temperature and read for agglutination after 15 minutes of incubation in accordance with standard procedures (22) . ...
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Background: Previous research strongly suggest that malaria is an important factor in the pathogenesis of endemic Burkitt’s lymphoma (eBL). Therefore, genetic factors such as sickle cell trait (SCT) and blood group-O that offer protection against severe malaria would be expected to reduce the risks of eBL. However, previous reports on the protective roles of SCT and blood group-O against the risks of eBL were inconclusive. Hence, the need for further studies on the protective roles of SCT and blood group-O separately, and also to investigate whether or not the combined anti-severe malaria protective roles of SCT and blood group-O have synergistic effects in reducing the risks of eBL. We therefore hypothesize that SCT and blood group-O are independently associated with reduced risks of eBL, and the co-inheritance of both factors (SCT and group-O) would provide greater protection against eBL. If our hypothesis is correct, children who inherited both SCT and blood group-O would have lower risks of eBL than their counterparts who inherited SCT or blood group-O separately. To the best of our knowledge, the possible synergistic relationship between SCT and blood group-O with regards to the risk of eBL has not been previously studied. Patients and Methods: We conducted a retrospective logistic regression analysis of the frequencies of Hb phenotypes and ABO blood groups among patients with eBL in order to determine the separate and synergistic protective effects of SCT and blood group-O on the risk of eBL in Nigeria where eBL is among the most common malignant childhood cancers. Results: The Odd Ratios (OR) for the risk of eBL were 0.52 for ‘SCT irrespective of ABO blood group’; 0.49 for ‘blood group-O irrespective of Hb phenotype’; and 0.23 for ‘SCT with blood group-O’. Discussion: These values suggest that both SCT and blood group-O are independently associated with modest reduction in the risk of eBL. However, when SCT with blood group-O was assessed for the risk factor for eBL, we obtained an Odds ratio of 0.23, which was significantly lower than the OR values for SCT (0.52) and blood group-O (0.49) separately. These figures suggest that coinheritance of SCT and blood group-O offers greater reduction in the risk of eBL than that provided by either SCT or blood group-O separately. The greater protection against eBL provided by the coinheritance of SCT and blood group-O is interpreted to be the resultant synergistic effect of the combined anti-malarial attributes of SCT and blood group-O. Conclusion: These findings suggest that the combined anti-malarial protective roles of SCT and blood group-O have synergistic effects in reducing the risks of eBL. This study has provided further evidence on the association between malaria-protective genetic polymorphisms and eBL, which is consistent with the aetiologic role of malaria in the pathogenesis of the tumour. Hence, the need for malaria endemic countries to intensify malaria control programs in order to curtail the incidence of eBL.
... Laboratory analysis: Method used for the determination of the percentage concentration of HbF was as earlier reported by Wild and Bain (2006). ...
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Hemoglobin F is normal hemoglobin seen in minute amount in adults. Increase in its level in adults is an indication of erythropoietic stress, which in most cases is linked to hemoglobinopathy. This study was undertaken to assess if physiological erythropoietic stress as seen in commercial blood donation, can increase it and thus be used as an indicator of frequency and duration of blood donation. The study involved 152 subjects including 88 commercial blood donors and 64 controls. Hemoglobin F was expressed as percentage concentration of the total hemoglobin. Results showed that hemoglobin F significantly increased in commercial blood donors when compared with the controls. There was also strong positive correlation between hemoglobin F level and age of the donors which was not the case with the controls. The results indicate that hemoglobin F level can be used as an indicator of the frequency and duration of blood donation. Though blood donation has some health benefits, the disadvantages of frequent donation outweigh these benefits and should be discouraged.
... Also, Reticulocytic count (RTX) was done for all students, by using Brilliant cresyl blue stain, to differentiate between types of hemolytic anemia and other types of anemia. [23] Also sickling test was done for all students for detecting the presence of HbS in SCD or SCT. All samples with evidence of anemia (Hb <11 g/dL and/or low MCV <68 fl) and/or high (RTX) and/or +ve sickling test were subjected to electrophoretic separation of (Hb-EPS) on cellulose acetate paper. ...
Article
ABSTRACT Iron deficiency anemia (IDA) is one of the most prevalent nutritional diseases in many parts of the world and the most common cause of anemia in KSA. Hemoglobin disorders as thalassaemia and sickle cell disease (SCD) have a big concern in many countries with high prevalence in KSA. The aim of this study was to determine the prevalence of IDA, sickle cell trait (SCT) and β-thalassaemia trait (βTT) among school children in Al-Khobar and Makkah cities, KSA. A multi stage random sample of 510 school children aged 12–18 years at both cities received health education about these disorders then investigated by complete blood count (CBC), serum iron, total iron binding capacity (TIBC), reticulocytic count and sickling test. Hemoglobin electrophoresis and high performance liquid chromatography (HPLC) were done in selected cases. Overall, the prevalence of IDA, SCT and βTT was 12.94%, 3.33% and 3.14% respectively with no significant difference between both cities. Significant variations were seen in the hematological parameters in students with IDA and βTT compared to normal students in each city but no significant differences in the hematological parameters between normal students and those with SCT. No significant differences were observed between both sexes for any of the blood characteristics examined except for iron and TIBC. Many factors contribute to the high prevalence of IDA, SCT and βTT in KSA including demographic factors, traditional and religious commonality and the high rate of consanguineous marriages in conjunction with genetic role. Strengthening public health education, nutritional programs and routine carrier screening for school children are recommended to help early discovery of different types of anemias. KEYWORDS: Prevalence, Iron deficiency anemia, Sickle cell trait, β-thalassaemia trait, School children.
... First, we recommend routine donor screening for SCT in Nigeria (and indeed other tropical regions in which the SCT is prevalent), as already advocated by the WHO 2 and recommended by local researchers.33 A simple and cheap HbS solubility test (rather than more costly Hb electrophoresis) would suffice as an effective and rapid SCT screening test in low resource tropical countries.36 Second, we recommend that blood banks and donation centres in Nigeria (and indeed other tropical regions in which the SCT is prevalent) should consider the possible application of a pre-donation hydration strategy for SCT-positive donors. ...
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Introduction: As a developing nation, Nigeria’s transfusion service is characterised by low quality control and a high incidence of clots in blood bags. About one-quarter of donors in Nigeria carry sickle cell trait (SCT), which is haemo-rheologically associated with pathological hyperviscosity and hypercoagulability. Hence, we hypothesised that SCT is a risk factor for clotting in bags, and that the risk would be aggravated by non-O blood groups due to their physiological (natural) hypercoagulability. Objectives: Determine risk of clotting in bags donated by persons with SCT and its interaction with ABO-blood groups, and proffer possible strategy to mitigate SCT-associated clotting risk in blood bags in Nigeria. Materials and Methods: Analysis of 100 clotted blood bags. Frequencies of SCT and ABO-groups of clotted and control bags were compared by Student-t and X2-tests. Risks of clotting for SCT with respect to ABO-groups were expressed as Odds ratios by case-control logistic regression. Results: Bags with ‘SCT and blood group-O’ had high clotting risk (OR=1.89, CI95%:1.43-2.34, p=0.031), while bags with ‘SCT and non-O groups’ had higher clotting risk (OR=2.97, CI95%:2.55-3.45, p=0.015); which suggested risk escalation by non-O groups. Discussion and Conclusion: SCT is independently associated with high clotting risk, which is synergistically escalated by additional hypercoagulable effects of non-O groups. Clots in blood bags have economic and clinical implications. Hence, blood banks in Nigeria (and other African countries with high prevalence of SCT) should upgrade quality control, routinely screen donors for SCT, and consider feasibility of pre-donation hydration for SCT-positive donors in order to reduce hyperviscosity and clotting risks in blood bags, thereby avoiding blood wastage and shortage
... (28) ABO blood groups (O, A, B, AB) and Hb phenotypes (HbAA, HbAS) were determined by monoclonal anti-sera and (29) and alkaline electrophoresis. (30) The types and number of spontaneous bleeding episodes were enumerated and collated with respect to the patients' ABO blood groups. The ABR for a patient refers to the number of spontaneous bleeding episodes in one complete year (12 consecutive calendar months). ...
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Background: Prothrombotic hypercoagulable conditions including sickle cell trait (SCT) and non-O blood groups have been independently associated with lower bleeding rates in haemophilia-A. Hence, we hypothesized that coinheritance of SCT and non-O blood groups would synergistically provide greater reduction in haemophilic bleeding rates. If our hypothesis is correct, severe haemophiliacs who co-inherited SCT and non-O blood groups would have lower annual bleeding rates (ABRs) than their counterparts who inherited SCT or non-O blood groups separately. Methods: To test our hypothesis, we conducted retrospective analysis of spontaneous ABRs with respect to coinheritance of haemoglobin (Hb) phenotypes and ABO blood groups among 84 severe haemophiliacs in four categories: haemophiliacs with coinheritance of HbAA and group-O (category1); haemophiliacs with coinheritance of SCT and group-O (category2); haemophiliacs with coinheritance of HbAA and non-O groups (category3); and haemophiliacs with coinheritance of SCT and non-O groups (category4). One-way ANOVA and Tukey‘s tests were used to determine statistical significance of variations in mean ABRs among the four categories. Results: Category1 had highest mean ABR of 47.8 (p < 0.05), because neither HbAA nor group-O had any hypercoagulability that can reduce bleeding rates. Category2 (mean ABR:34.1) and category3 (mean ABR:32.3) had intermediate and comparable mean ABRs (p>0.05), suggesting that SCT and non-O groups are independently associated with comparable but modest reductions in bleeding rates. Category4 had lowest mean ABR of 25.3 (p<0.05), which reflects synergistic effects of SCT and non-O groups. Conclusion: These results suggest that SCT and non-O blood groups synergistically provided greater reduction in bleeding rates. Hence, coinheritance of Hb phenotypes and ABO blood groups may modify haemophilic bleeding phenotypes in Africans and other populations in which the sickle cell mutation is prevalent. Larger studies are needed to validate these preliminary findings.
... The highest frequency of sickle cell gene in India is reported in Orissa (9%), followed by Assam (8.3%), Madhya Pradesh (7.4%), Uttar Pradesh (7.1%), Tamil Nadu (7.1%) and Gujarat (6.4%). 9,10,11 The high incidence of sickle cell trait requires need of premarital counseling and also antenatal screening to prevent sickle cell disease in off springs. ...
... Hb C, the second most common structural Hb variant in people of African descent, is caused by the substitution of glutamic acid in position 6 of the β chain by lysine (β6Glu → Lys). [11] Some vaso-occlusive complications accompanying the inheritance of abnormal Hb variants include splenic infarction and attendant autosplenectomy, stroke, bone infarcts and aseptic necrosis, leg ulcers, priapism, pulmonary hypertension, pyelonephritis, pulmonary infarction, pneumonia, acute chest syndrome, prematurity, and renal failure. The severity of the clinical manifestation varies greatly from patient to patient influencing the aggressiveness of treatment modification accordingly. ...
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Background: Attempts being made to unravel the relationship between blood antigens, hemoglobin (Hb) genotypes and increased susceptibility to certain diseases are ongoing. Objective: The study was carried out to provide data on the distribution of ABO, Rh D, and Hb variants among pregnant women receiving antenatal care at Federal Medical Centre, Yola, Nigeria. Materials and Methods: Medical records of these subjects (n = 2226) were sorted out and analyzed according to their ABO blood group phenotypes and Hb variants. Results: Blood group O was the most prevalent (47.7%) among our subjects, followed by A (26.6%), B (22.2%), and AB (3.5%). The Rh D antigen was positive in 97.1% and negative in 2.9% of the study population. There were five Hb genotypes AA (81.94%), AC (0.34%), AS (17.57%), SC (0.05%), and SS (0.14%). The occurrence of different ABO, Rh D blood groups and Hb variants varied significantly (P < 0.05) among pregnant women studied. Conclusion: The frequency of ABO and Rh D blood groups will assist in the formulation of genetic counseling policies.
... 3 Cation exchange high-performance liquid chromatography (CE-HPLC) has to lead to a simple rapid and superior method for detection and quantification of these variants, in routine laboratory practice. 4 Detection of asymptomatic carriers and double heterozygous states are the most important advantages of this method. Early, accurate detection of carriers can aid in prevention and management of various hemoglobinopathies. ...
... On the basis of the electrophoretic patterns, phenotypes were categorized as normal (HbAA) or SCT (Hb AS). (16) A variety of organ damage that were diagnosed on the basis of documented clinical, laboratory and radiological evidences at the time of diagnosis of DIC were identified and enumerated. In this study, acute renal injury was diagnosed if the level of serum creatinine was elevated above 130 μmol/L (17) ; acute hepatic injury was diagnosed if the levels of hepatic transaminases were elevated above 35 IU/L for alanine aminotransferase (ALT) and above 42 IU/L for aspartate aminotransferase (AST) (17) ; acute pulmonary injury was diagnosed if there were documented clinical features of respiratory distress and a pulse oxymetry SpO2 level lower than 95% with associated pulmonary opacities on chest radiographs (18) ; and acute thrombotic musculoskeletal injury was diagnosed if there were documentations of gangrenous lesions in one or more toes or fingers or any part of patient limbs characterized by blackish discoloration of the overlying skin with radiological evidence of necrosis of the underlying tissues. ...
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Background: Disseminated intravascular coagulation (DIC) is associated with fibrin deposition, tissue hypoxia and multi-organ damage (MOD). We believe that hypoxia due to fibrin deposition would trigger red cell sickling and aggravate risk of MOD among DIC patients with sickle cell trait (SCT). Hence, we hypothesize that red cell sickling could be a co-factor in pathogenesis of MOD in DIC patients with SCT, and we predict that SCT (HbAS phenotype) would be a risk factor for MOD in DIC. If our hypothesis and prediction are correct, DIC patients with SCT will have higher frequency of organ damage when compared with their counterparts with normal (HbAA) phenotype. Materials and Methods: We performed a retrospective comparative analysis of frequencies of organ damage with respect to Hb-phenotypes (SCT vs. HbAA) among DIC patients in Nigeria where SCT is prevalent in up to one-third of the population. Results: In comparison with their HbAA counterparts, DIC patients with SCT had higher frequency of organ damage (50% vs. 14.1%, p<0.05) with a corresponding elevated relative risk of organ damage (RR=2.3, CI95%: 1.8-3.5, p=0.005). Conclusion: This study suggest that the dual effect of fibrin deposition and red cell sickling increased the vulnerability of DIC patients with SCT to severe microvascular occlusion, greater hypoxia, and higher risk of MOD. Conversely, DIC patients with HbAA phenotype sustained microvascular blockade due only to the effect of fibrin deposition resulting in a relatively lower risk of MOD. This preliminary report suggests that SCT is a risk factor for MOD in DIC. There is however, the need for larger prospective study to confirm the validity of this report. Meanwhile, we recommend that DIC patients with SCT need closer monitoring and whenever possible, DIC patients with SCT who require transfusion should be transfused with red cells obtained from donors with normal HbAA phenotype.
... El diagnóstico de un trastorno de hemoglobina requiere de una combinación de técnicas de laboratorio, que deben ser interpretadas en el contexto de la edad y sexo del paciente, de su historia personal, de sus antecedentes familiares y de su origen étnico. 12 El estudio de laboratorio incluye los siguientes métodos: 12,13,[20][21][22][23] H e m o g r a m a : L a h e m o g l o b i n a e s t á , generalmente, 2-3 g/dL por debajo del valor normal, aunque puede ser normal. El recuento de eritrocitos es elevado, mientras que el VCM y la HCM están disminuidos. ...
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Hypochromic microcytic anemias are the most frequent anemias in pediatric patients. The majority of cases are secondary to iron deficiency, but hemoglobin disorders, mainly thalassemia minor, must always be taken into consideration in Argentina. Hereditary disorders of iron metabolism, in spite of their very low prevalence, have to be borne in mind too. This guide establishes criteria for the differential diagnosis between these anemias, with a double aim: a) to facilitate starting treatment as soon as possible; and b) to give adequate genetic counseling regarding hereditary diseases.
... Detection of hemoglobinopathies is possible by a number of techniques like hemoglobin electrophoresis, HPLC, isoelectric focusing, capillary gel electrophoresis and molecular analysis. [4] With HPLC, quantification of different hemoglobin subtypes is possible in a single and highly reproducible system. It is an easy to perform test and can replace laborious procedures like estimation of HbF and HbA2. ...
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Background: High performance liquid chromatography (HPLC) is the most commonly used method for detection and quantitative estimation of hemoglobin variants. Hemoglobinopathies are amongst the most common genetically inherited disorders, however, the exact magnitude of different hemoglobinopathies is obscure in India. This study was done with the aim of analyzing the different findings in HPLC using D-10 analyzer and evaluating the spectrum of different hemoglobin disorders in a hospital-based population of South Delhi. Such a prevalence study would be useful to review the various strategies that can be implemented for effective control and prevention of these disorders. Methods: A hospital based descriptive observational study was conducted in which all OPD and IPD patients who were advised HPLC during their clinical workup were included. Analysis of EDTA blood samples was done by Bio Rad D10 Dual program HPLC instrument. The exact percentage of HbA, HbA2, HbF and any other variant hemoglobin was estimated. Presumptive identification of hemoglobin variants was made primarily by their percentage, retention time (RT) and peak characteristics. HPLC findings were correlated with the clinical history, family history and the CBC and peripheral smear findings in all cases. Results: On HPLC analysis, 79% of the patients had no abnormality detected and the report was within normal limits. The commonest hemoglobinopathy was Beta Thalassemia Trait followed by HbE trait. The other hemoglobinopathies detected were HbD Punjab Heterozygous (3 cases, 0.5%), Beta thalassemia homozygous (3 cases, 0.5%), Sickle cell Heterozygous (2 cases, 0.3%), HbJ Meerut Heterozygous (2 cases, 0.3%). One case each of Sickle cell Homozygous (0.15%), Compound Heterozygous HbS/beta thalassemia trait (0.15%), HbE Homozygous (0.15%), Compound Heterozygous HbE/beta thalassemia trait (0.15%), and Homozygous delta beta thalassemia (0.15%) were also diagnosed. Conclusion: This study gives an important insight to the present day scenario of hemoglobinopathies in patients in South Delhi in relation to the hematological profile. It highlights the chromatogram findings of different hemoglobinopathies on the D10 analyzer. The comprehensive data obtained by such series can help in the formulation and development of infrastructure and policies for hemoglobinopathy prevention, diagnosis and management.
... Although there are contradicting results on the impact of HU on VTE prevention, these current results were in inconclusive with respect to a positive role of HU in VTE prevention, knowing that that HbF inhibits intracellular HbS polymerization. 37,38 In the current study population, there were elevations in the median WBC count, LDH level, bilirubin and CRP in the case group from baseline to VTE episode; and the baseline levels were significantly different compared with the control group. Higher WBC count and elevated LDH level are reported to be risk factors for VTE in SCA. ...
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Objective To assess the clinical and laboratory predictors of venous thromboembolism (VTE) in patients with sickle cell anaemia (SCA) and its relationship to morbidity and mortality. Methods This retrospective case–control study analysed data from patients with SCA that experienced VTE compared with matched control patients with SCA but no VTE (2:1 ratio). Results A total of 102 patients with SCA were enrolled (68 cases with VTE and 34 controls). Amongst the 68 cases (median age, 29.5 years), 26 (38.2%) presented with isolated pulmonary embolism (PE). A higher prevalence of splenectomy (73.5% versus 35.3%) was observed in the cases compared with the controls. A significantly higher prevalence of central venous catheter (CVC) insertion (42.6% versus 8.8%) was observed in the cases compared with the controls. High white blood cell counts, serum lactic dehydrogenase (LDH), bilirubin and C-reactive protein (CRP) and low haemoglobin (Hb) and HbF were significant risk factors for VTE. Forty-two cases (61.8%) developed acute chest syndrome, 10 (14.7%) had a stroke and seven (10.3%) died. Conclusions VTE in patients with SCA has a high impact on morbidity and mortality. PE was the leading presentation of VTE, with CVC insertion, high LDH, bilirubin, CRP and white blood cell counts along with low Hb and HbF constituting other significant risk factors.
... The highest frequency of sickle cell gene in India is reported in Orissa (9%), followed by Assam (8.3%), Madhya Pradesh (7.4%), Uttar Pradesh (7.1%), Tamil Nadu (7.1%) and Gujarat(6.4%) [13][14][15] . But in light of population migration it is becoming a worldwide phenomenon. ...
Article
Introduction: Sickle cell haemoglobinopathy is an inherited hemoglobinopathy resulting from a mutation occurring in betaglobin gene, on chromosome 11. The gene is prevalent in some tribes of North Maharashtra. The main aim of the study is to determine haemoglobin patterns in cases with sickle cell hemoglobinoathies in North Maharashtra using HPLC testing system. Material and Methods: This is a prospective study done over a period of 6 years. 10081 patients having positive solubility test or negative solubility test but having clinical suspicion of Sickle cell hemoglobinopathies were studied in detail and all samples were subjected for HPLC testing. Results: Prevalence of sickle cell hemoglobinopathy in this study was 70.36%. Most common pattern of haemoglobin observed was SA (89.72%). A slight female preponderance (54%) was noted. Predominant age group was paediatric (39.96%), followed by12-20yrs (33.97%). Oldest case for HbSS was 55yrs male. Predominant category affected was ST (82.05%). Conclusion: A very high prevalence of Sickle cell hemoglobinopathy was noted in this study. This is because the study was done in areas where Pawara and Bhill community resides who have a high frequency of HbS gene. Solubility test was found to be cost effective and easy screening test (Sensitivity being70.36%). HPLC found to be Rapid and accurate test for diagnosis of hemoglobinopathy and had helped in diagnosis of some rare heterozygous disorders like SA-HBQ India, SA-Hereditary persistence of foetal haemoglobin, HBD-SA. This is one of the largest and first of its kind prospective study which will help in prevention and cost effective management in targeted population.
... Aliquots of venous blood samples from each study participant were separately submitted for routine haematological assays, which included total leukocytes, platelet and reticulocyte counts using standard haematological methods. (21,23,24) Blood Hb level was determined using Drabkin's reagent at 540 nm. (25) Each assay was run in duplicate. ...
... In practice however, most clinics in developing countries rely mainly on conjunctival inspection. [32] During pregnancy, efforts should be geared toward the early detection and treatment of anaemia before delivery. Also, medical staff managing pregnant women should endeavour to investigate anaemic pregnant women further in order to identify the etiology of anaemia whenever possible, despite commencing the usual treatment with iron and folate. ...
... Laboratory analysis: Method used for the determination of the percentage concentration of HbF was as earlier reported by Wild and Bain (2006). ...
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Hemoglobin F is normal hemoglobin seen in minute amount in adults. Increase in its level in adults is an indication of erythropoietic stress, which in most cases is linked to hemoglobinopathy. This study was undertaken to assess if physiological erythropoietic stress, as seen in commercial blood donation, can increase it and thus be used as an indicator of frequency and duration of blood donation. The study involved 152 subjects including 88 commercial blood donors and 64 controls. Hemoglobin F was expressed as percentage concentration of the total hemoglobin. Results showed that hemoglobin F significantly increased in commercial blood donors when compared with the controls. There was also strong positive correlation between hemoglobin F level and age of the donors which was not the case with the controls. The results indicate that hemoglobin F level can be used as an indicator of the frequency and duration of blood donation. Though blood donation has some health benefits, the disadvantages of frequent donation outweigh these benefits and should be discouraged.
... Hb phenotypes were determined by haemoglobin electrophoresis at a pH of 8.6 on cellulose acetate paper, sickling test and haemoglobin quantitation. On the basis of the electrophoretic patterns, patients were categorised as normal (Hb AA phenotype) or SCT (Hb AS phenotype) [22]. ...
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Haemophilia A is an X-linked recessive disorder associated with deficiency of coagulation factor VIII and lifelong bleeding diathesis. Sickle cell trait (SCT) is the heterozygous state for the sickle β-globin gene. The frequency of SCT is up to 30% in Africa, wherein it confers survival advantage by providing resistance against severe malaria. SCT does not cause vaso-occlusive crisis, but is associated with high risk of venous thromboembolism as variously reported in the literature. We consider SCT as a hypercoagulable prothrombotic state and hypothesise that coinheritance of SCT may ameliorate the clinical phenotype of severe haemophilia. We conducted a retrospective analysis of frequencies of spontaneous bleeding among severe haemophiliacs with SCT (Hb AS phenotype) and their counterparts with normal Hb phenotype (Hb AA phenotype) in order to determine the possible ameliorating effect of SCT on spontaneous bleeding rates in severe haemophilia A. If our hypothesis is correct, severe haemophiliacs with SCT will have lower frequencies of spontaneous bleeding than their counterparts with normal Hb phenotype. Our results revealed that severe haemophiliacs with normal Hb phenotype had significantly higher mean annual bleeding episodes per patient in comparison with their counterparts with SCT (45±7 vs 31±5, p=0.033), suggesting that severe haemophiliacs with SCT had lower frequencies of spontaneous bleeding episodes. The result of this study indicates that coinheritance of SCT in patients with severe haemophilia may be associated with reduced frequency of spontaneous bleeding, which may imply better overall prognosis. However, the study has important limitations, which include its retrospective nature and the very low number of subjects. The findings should therefore be validated by a larger and prospective study.
... Methaemoglobin levels vary but may be as high as 40% of the total haemoglobin [8]. The haematological value for normal adults expressed as a mean ±2SD (95% range) is less than 2% [9]. ...
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The first case of methaemoglobinaemia was reported by Sloss and Wybauw in 1912. Methaemoglobin is a derivative of normal haemoglobin in which the iron of the haem complex has been oxidized from the ferrous to the ferric form. Methaemoglobin does not combine with oxygen and thus does not take part in oxygen transport. In normal red cells, methaemoglobin is continually being formed by the auto-oxidation of haemoglobin, but it is reduced as soon as it is formed; thus the concentration of methaemoglobin in the red blood cells under normal conditions is less than one percent of total haemoglobin. The term methaemoglobinaemia is used to describe the excess accumulation of methaemoglobin in the red blood cell. Methaemoglobin lacks the capacity to carry oxygen, and methaemoglobinaemia causes symptoms and signs of hypoxia. Toxic methaemoglobinaemia occurs when a drug or other toxic substance oxidizes haemoglobin and the patient is likely to show cyanosis. Methaemoglobinaemia has a wide spread among industrial workers worldwide where they make use of toxic chemicals, also among fertilizer company workers. Methaemoglobin can be identified spectroscopically by its absorption band in the red part of the spectrum at 630nm; this band disappears on the addition of yellow ammonium sulphide. Methaemoglobin levels are best measured using the change of absorbance of methaemoglobin of 630nm that occurs when cyanide is added, converting the methaemoglobin to cyanmethaemoglobin. Treatment consists of slow intravenous injection of methylene blue; the recommended does is 2mg/kg bodyweight for infants, 1.5mg/kg bodyweight for older children, and 1mg/kg bodyweight for adults, in a 1% sterile aqueous solution.
... On the basis of the electrophoretic patterns, phenotypes were categorized as normal (HbAA) or SCT (Hb AS). (16) A variety of organ damage that were diagnosed on the basis of documented clinical, laboratory and radiological evidences at the time of diagnosis of DIC were identified and enumerated. In this study, acute renal injury was diagnosed if the level of serum creatinine was elevated above 130 μmol/L (17) ; acute hepatic injury was diagnosed if the levels of hepatic transaminases were elevated above 35 IU/L for alanine aminotransferase (ALT) and above 42 IU/L for aspartate aminotransferase (AST) (17) ; acute pulmonary injury was diagnosed if there were documented clinical features of respiratory distress and a pulse oxymetry SpO2 level lower than 95% with associated pulmonary opacities on chest radiographs (18) ; and acute thrombotic musculoskeletal injury was diagnosed if there were documentations of gangrenous lesions in one or more toes or fingers or any part of patient limbs characterized by blackish discoloration of the overlying skin with radiological evidence of necrosis of the underlying tissues. ...
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Background: Disseminated intravascular coagulation (DIC) is associated with fibrin deposition, tissue hypoxia and multi-organ damage (MOD). We believe that hypoxia due to fibrin deposition would trigger red cell sickling and aggravate risk of MOD among DIC patients with sickle cell trait (SCT). Hence, we hypothesize that red cell sickling could be a co-factor in pathogenesis of MOD in DIC patients with SCT, and we predict that SCT (HbAS phenotype) would be a risk factor for MOD in DIC. If our hypothesis and prediction are correct, DIC patients with SCT will have higher frequency of organ damage when compared with their counterparts with normal (HbAA) phenotype. Materials and Methods: We performed a retrospective comparative analysis of frequencies of organ damage with respect to Hb-phenotypes (SCT vs. HbAA) among DIC patients in Nigeria where SCT is prevalent in up to one-third of the population. Results: In comparison with their HbAA counterparts, DIC patients with SCT had higher frequency of organ damage (50% vs. 14.1%, p<0.05) with a corresponding elevated relative risk of organ damage (RR=2.3, CI95%: 1.8-3.5, p=0.005). Conclusion: This study suggest that the dual effect of fibrin deposition and red cell sickling increased the vulnerability of DIC patients with SCT to severe microvascular occlusion, greater hypoxia, and higher risk of MOD. Conversely, DIC patients with HbAA phenotype sustained microvascular blockade due only to the effect of fibrin deposition resulting in a relatively lower risk of MOD. This preliminary report suggests that SCT is a risk factor for MOD in DIC. There is however, the need for larger prospective study to confirm the validity of this report. Meanwhile, we recommend that DIC patients with SCT need closer monitoring and whenever possible, DIC patients with SCT who require transfusion should be transfused with red cells obtained from donors with normal HbAA phenotype.
... The prospective couples have to contact the designated center, where trained staff (usually pediatricians, but if not available, a designated physician) will record their basic demographic information and collect the blood samples into tubes (containing ethylenediamine tetraacetic acid anticoagulant) for testing at the designated laboratory. Hemoglobinopathies are identified via hemoglobin electrophoresis, and the results are interpreted in accordance with standard laboratory diagnostic protocols [12]. ...
... Patients were categorised as having severe, moderate or mild haemophilia [19] . ABO blood groups (O, A, B, AB) and Hb phenotypes (HbAA or HbAS) were determined by routine methods using monoclonal anti-sera and alkaline electrophoresis [20,21] . ...
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Background: In tropical countries such as Nigeria, where factor VIII (FVIII) is scarce, severe pain due to musculoskeletal bleeding complications, leading to frequent opioid prescription, is not uncommon in poorly managed people with haemophilia (PWH). The relationship between opioid use and dependence is intensively studied in other painful diseases, such as cancer and rheumatoid arthritis, but surprisingly little is known about opioid dependence in haemophilia. We hypothesise that the risk of opioid dependence among PWH in tropical countries like Nigeria is multi-factorial, encompassing demographic (age), clinical (haemophilia severity and chronic arthropathy) and biological (ABO blood groups and haemoglobin (Hb) phenotypes) factors that may directly or indirectly increase incidence of bleeding and/or pain. Aims: To determine the prevalence of opioid dependence and relative risks (RR) associated with age, haemophilia severity, chronic arthropathy, ABO blood groups and Hb phenotypes, and to elucidate the pathophysiological roles of each risk factor in the development of opioid dependence among haemophilia-A patients in five hospitals in northern Nigeria. Methodology: A retrospective review of the medical records of 88 PWH seen between 1996 and 2012 was used to collate data on age, sex, haemophilia severity, painful chronic haemophilic arthropathy, ABO blood group, haemoglobin phenotypes, presence or absence of opioid dependence, and the types of opioids on which the patients were dependent. The prevalence of opioid dependence among the cohort was expressed as a percentage. The frequency of each putative risk factor for opioid dependence in patients with and without opioid dependence were compared using Fisher’s exact test; RR associated with each risk factor was determined by regression analysis. P<0.05 was taken as significant. Results: Of the 88 PWH studied,15 (17%) were shown to be opioid-dependent. Compared with PWH who were not opioid-dependent, this group had higher frequencies of severe haemophilia (86.7% vs. 49.3%: RR= 5.2, p=0.02), survival to adulthood (73.3% vs. 12.3%: RR= 9.5, p=0.0001), chronic arthropathy in one or more joints (86.7% vs. 21.9%: RR= 13.2, p=0.0004), blood group-O (80% vs. 49.3%: RR= 3.3, p=0.04), and HbAA phenotype (86.7% vs. 54.8%: RR= 4.3, p=0.04). Conclusion: Prevalence of opioid dependence among PWH treated at five hospitals in northern Nigeria was 17% during the study period. Significant risk factors were directly or indirectly associated with increased rates of bleeding and/or pain, which can only be prevented or treated through optimal application of FVIII. There is a need for the Nigerian government to establish standard haemophilia care centres with adequate FVIII for optimal prophylaxis and treatment in order to minimise painful complications, thereby helping to prevent undue opioid use and dependence.
... (28) ABO blood groups (O, A, B, AB) and Hb phenotypes (HbAA, HbAS) were determined by monoclonal anti-sera and (29) and alkaline electrophoresis. (30) The types and number of spontaneous bleeding episodes were enumerated and collated with respect to the patients' ABO blood groups. The ABR for a patient refers to the number of spontaneous bleeding episodes in one complete year (12 consecutive calendar months). ...
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Background: Prothrombotic hypercoagulable conditions including sickle cell trait (SCT) and non-O blood groups have been independently associated with lower bleeding rates in haemophilia-A. Hence, we hypothesized that coinheritance of SCT and non-O blood groups would synergistically provide greater reduction in haemophilic bleeding rates. If our hypothesis is correct, severe haemophiliacs who co-inherited SCT and non-O blood groups would have lower annual bleeding rates (ABRs) than their counterparts who inherited SCT or non-O blood groups separately. Methods: To test our hypothesis, we conducted retrospective analysis of spontaneous ABRs with respect to coinheritance of haemoglobin (Hb) phenotypes and ABO blood groups among 84 severe haemophiliacs in four categories: haemophiliacs with coinheritance of HbAA and group-O (category1); haemophiliacs with coinheritance of SCT and group-O (category2); haemophiliacs with coinheritance of HbAA and non-O groups (category3); and haemophiliacs with coinheritance of SCT and non-O groups (category4). One-way ANOVA and Tukey‘s tests were used to determine statistical significance of variations in mean ABRs among the four categories. Results: Category1 had highest mean ABR of 47.8 (p < 0.05), because neither HbAA nor group-O had any hypercoagulability that can reduce bleeding rates. Category2 (mean ABR:34.1) and category3 (mean ABR:32.3) had intermediate and comparable mean ABRs (p>0.05), suggesting that SCT and non-O groups are independently associated with comparable but modest reductions in bleeding rates. Category4 had lowest mean ABR of 25.3 (p<0.05), which reflects synergistic effects of SCT and non-O groups. Conclusion: These results suggest that SCT and non-O blood groups synergistically provided greater reduction in bleeding rates. Hence, coinheritance of Hb phenotypes and ABO blood groups may modify haemophilic bleeding phenotypes in Africans and other populations in which the sickle cell mutation is prevalent. Larger studies are needed to validate these preliminary findings.
... 20 ABO blood groups (O, A, B, AB) and Hb phenotypes (HbAA or HbAS) were determined by routine manual methods using monoclonal anti-sera and alkaline electrophoresis. 21,22 Parasitological diagnosis of malaria, identification of bleeding episodes, platelet count: The standard of care for febrile haemophilia patients in the centres of this study included general clinical evaluation, malaria diagnostic evaluations by light microscopy 23 , physical assessment of any bleeding episodes, and platelet count by automation or standard manual techniques. 24 Diagnosis and assessment of malaria infection were based on microscopic examination of Giemsa stained thick blood smears and Leishman stained thin blood smears using standard techniques. ...
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Malarial thrombocytopenia (MT) and its haemorrhagic effect on haemophiliacs have not been studied in Nigeria. We hypothesized that the risk of malarial thrombocytopenic bleeding (MTB) among haemophiliacs with MT in Nigeria would be affected by demographic and clinical factors that may affect platelet function, host susceptibility to malaria or its clinical severity. To determine incidence, pattern, demographic and clinical risk factors of MTB among haemophiliacs with MT, we retrospectively studied 95 haemophiliacs with MT with respect to MTB, age, sex, severity of haemophilia, thrombocytopenia, parasitemia, haemoglobin (Hb) phenotypes and ABO blood groups. Relative risk (RR) of MTB for each parameter was calculated. Out of 95 haemophiliacs with MT, 16(16.8%) had muco-cutaneous MTB. No significant difference in frequency of severe haemophilia between haemophiliacs with and without MTB (50% vs.48.1%, RR=1.1, p=0.89). But haemophiliacs with MTB had higher frequency of age <5years (81.3% vs. 38.0%: RR=5.2, p=0.006), higher frequency of platelet count <20x10 9 /L (81.3% vs. 21.5%, RR=9.4, p=0.0002), higher frequency of malaria parasite density ≥3+ (68.7% vs. 11.4%, RR=8.3, p=0.0001), higher frequency of HbAA phenotype (75% vs. 44.3%, RR=3.1, p=0.04), and higher frequency of non-O blood groups (62.5% vs. 32.9%, RR=2.7, p=0.03). In conclusion, incidence of MTB among haemophiliacs with MT was 16.8%. Risk of MTB was not affected by severity of haemophilia, but the risk was increased by young age, severity of parasitemia and thrombocytopenia, non-O blood group and HbAA phenotype. Therefore, haemophiliacs presenting with triad of fever, thrombocytopenia and muco-cutaneous bleeding in the tropics should be investigated for malaria.
... Equivocal results were those in which there was a very fine cloudiness in the test tube and the edges of the lines slightly blurred. The control sample must be fully lysed; if not the result of test was considered invalid [15,16,17]. Only one control was performed with the batch of tests per day. ...
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Background: Thalassaemia is highly prevalent in Sri Lanka. The highest number of patients are seen in the North Western province, especially in the Kurunegala district. Screening of children and adults to detect beta thalassaemia carriers/trait using automated full blood counts and high performance liquid chromatography (HPLC) are only limited to few centers in Sri Lanka. In this context, the value of one tube osmotic fragility test (OFT) as a screening test is immense. Our study tried to determine the sensitivity and specificity of OFT in beta thalassaemia trait (BTT) in Sri Lanka. Materials and Methods: This was a cross sectional study on randomly selected 700 subjects, carried out at National Thalassaemia Center (NTC), Kurunegala. Participants were categorized into four groups based on red cell indices, HPLC and serum ferritin assay as normal group, BTT group, iron deficiency anaemia (IDA) group and other haemoglobinopathies. OFT was performed on all cases. Original Research Article Pilapitiya et al.; AJMPCP, 4(1): 7-13, 2021; Article no.AJMPCP.64291 8 Results: Out of 700 subjects 396 subjects (56.6%) were females and 304(43.4%) were males. OFT gave definitely positive or equivocal results in 194 of 201 patients with BTT and 96.52% of sensitivity was observed. The test was false positive in 2 out of 268 (0.75%) normal subjects and 99.25% of specificity was observed. There were 3.48% (7/201) false negative results with a negative predictive value (NPV) of 97.44% and the positive predictive value (PPV) of the test was 98.97%. Conclusion: One tube osmotic fragility test is a sensitive, cost effective, rapid and reliable primary screening test for detection of BTT in a population with financial restrictions.
... Beta thalassemia and related hemoglobinopathies are diagnosed by a number of techniques including hemoglobin electrophoresis, high performance liquid chromatography (HPLC), iso-electric focusing, capillary gel electrophoresis and molecular analysis. 3 High performance liquid chromatography (HPLC) is universally accepted as the most useful initial screening method for thalassemia and other hemoglobinopathy detection. 4 The conventional HPLC instrument (VARIANT HEMOGLOBIN TESTING SYSTEM, BIO-RAD) although well standardized, is very costly (more than Rs. ...
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Background: Hemoglobinopathies especially thalassaemia and its interaction with HbE and HbS are significant cause of morbidity and mortality in our country. With no feasible treatment, prevention of cases by carrier detection is the only option for successful reduction of the disease burden. VARIANT hemoglobin testing system BIORAD using principle of cation exchange chromatography has been in use and considered as gold standard for carrier detection. The purpose of this study is to compare the efficacy of a different, cheaper instrument; D10 by the same manufacturer BIORAD for carrier detection in beta thalassaemia.Methods: Patients diagnosed as beta thalassaemia carrier by VARIANT hemoglobin testing system (HbA2 value between 4.0-9.0) were retested using D10 instrument and checked for agreement.Results: There was good correlation between VARIANT and D-10 methods with Intraclass correlation coefficient 0.832 (95% Confidence Interval 0.756-0.884). Bland-Altman analysis showed mean bias of +0.3526 (95% CI -0.3958 to +1.101).Conclusions: Although further study is needed with larger sample size for assessment of sensitivity and specificity of D10 instrument, it is evident from this study that this instrument can be an effective and cheaper alternative of VARIANT hemoglobin testing system.
... Criteria for IDA was considered in anemic pupils with TS > 5% [13] , or 5% SF > 15 ng/ml in those having TS equal or more than 5% [14] . Hb A 2 above 3.7% was considering the diagnosis of -thalasemia trait [15] . ...
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Objective: To find out the prevalence of iron deficiency anemia (IDA) among adolescents intermediate school pupils. Subjects & Methods: A study was carried out on 520 (320 boys and 200 girls) intermediate school pupils. Boys of less than 15 years old and girls were characterized anemic if their packed cell volume (PCV) were < 36% , and < 39 in boys of 15 years old or above. Iron deficiency anemia was considered if anemic pupil shows TS < 5% or SF < 15% when TS were 5% or above. Results: The prevalence of IDA was found to be 20.35%, with a higher prevalence in girls than in boys. It is mostly noticed at age of 15 in girls and 17 in boys. IDA was diagnosed by TS > 5% in 56.19% pupils and low SF in 43.81%. Conclusion: This study clarified that IDA is a health problem of moderate severity among adolescent intermediate school pupils in Ramadi district. To overcome this health problem we recommend to detect and treat the anemic pupils in addition to ensure them about the iron rich diet are important measures.
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The study aimed at evaluating the variability in some haemo-rheological parameters of individuals having different haemoglobin genotypes in Rumuche community, Emohua Local Government Area of Rivers state. A cross-sectional and comparative study design was applied. A total of eighty-seven subjects (sixty-nine AA and eighteen AS haemoglobin genotypes) aged between 20-90 years were recruited. Samples collected were analyzed for packed cell volume, erythrocyte sedimentation rate, haemoglobin and fibrinogen concentration using standard methods. Results for haemoglobin genotypes reveals that sixty-nine subjects were haeamoglobin AA (79.31 %), eighteen subjects were haemoglobin AS (20.69 %), no haemoglobin SS genotype was identified among the subjects. Also, the result showed a significant difference in haemoglobin concentration when haemoglobin AA were compared to AS genotypes (14.68 ± 2.600g/dl vs 12.76 ± 2.931g/dl; p = 0.0450). Packed cell volume, erythrocyte sedimentation rate and fibrinogen concentration showed no statistical difference despite variations upon comparison of haemoglobin AA and haemoglobin AS. Based on gender, the variations observed were not statistically significant in all parameters at p < 0.05. The study revealed a significant increase in haemoglobin concentration in favour of individuals with haemoglobin AA genotype. Therefore, a directly proportional difference in the blood viscosity of the different haemoglobin genotypes was observed and this can be a useful diagnostic tool alongside fibrinogen concentration and packed cell volume for predicting the occurrence of some diseases such as atherosclerosis, thrombotic disorders, cardiovascular diseases or haemorrhagic disorders. No variation was observed in packed cell volume, erythrocyte sedimentation rate, and fibrinogen.
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p> Back ground: Iron deficiency anemia (IDA) and beta-thalassaemia trait (B-TT) are the most common causes of hypochromic microcytic anemia. Many indices have been defined to quickly discriminate these similar entities via parameters obtained from automated blood count analyzers. Methodology: The purpose of the study was to evaluate the predictive value of these indices in differential diagnosis of IDA and B-TT in adult cases. In this study we use auto-analyzer based formula of percentages of microcytic and hypochromic red cells (M/H ratio=% of Microcytosis/% of Hypochromia) as a screening tool for thalassaemia trait in Bangladeshi population. Results: A total of 150 subjects w ere included in this study with 50 known obligate carrier of beta-thalassaemia trait and 100 patients with hypochromia and microcytosis. Confirmatory tests for IDA were performed if serum ferritin level was <12 ng/ml and confirmatory test for Beta-thalassaemia trait (BTT) and Haemoglobin E trait can be considered when HbA2 > 3.5% , on agarose gel Haemoglobin electrophoresis at an alkaline pH (8.6) where, in addition, MCV <76 fl and/or MCH <27 pg. BTT was selected with HbA2 >3.5%, while the non- BTT group were those with HbA2 <3.5%.The final analysis of the result revealed that M/H ratio is a very sensitive index for betathalassaemia trait. In our study, the sensitivity, predictive value and diagnostic accuracy of the M/H ratio for the beta thalassaemia trait were 96%, 90.4% and 90.4% respectively and also identify all cases of coexistent iron deficiency. Conclusion: Thus M/H ratio is an easy, reliable and sensitive index which can be used for mass screening of betathalassaemia trait, particularly in a population where iron deficiency anemia is also prevalent. Anwer Khan Modern Medical College Journal Vol. 7, No. 1: Jan 2016, P 5-10</p
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p class="Abstract">A 5½ year old male child of a consanguineous couple presented with moderately high fever, hepatosplenomegaly and severe pain in the left upper quadrant of the abdomen for four days. On examination, the child was found severely anemic, ill-looking and mildly icteric but the abdomen was soft and non-tender. Radiological investigations and ultrasonography of the whole abdomen revealed the diagnosis of space occupying lesions which may be either due to splenic abscess/cyst or leukemia/lymphoma. However, blood film revealed the features of hereditary hemolytic anemia and later on hemoglobin electrophoresis initially commented as HbS–beta thalassemia but subsequent family screening of the patient turned out to a case of homozygous sickle cell anemia or sickle cell disease. To resolve such diagnostic dilemma, it is very much essential to analyze critically the history including family history, clinical and physical findings as well as investigational findings.</p
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Extended spectrum beta-lactamases (ESBL) produced by Gram-negative uropathogens constitute an emerging public-health concern. The aim of the study was to determine the prevalence of ESBL producing enterobacteriaceae uropathogens in Sokoto. The study was undertaken at Specialist Hospital Sokoto (SHS) over a 2-month period. Ninety-one urine samples were collected. The isolates collected were identified using biochemical tests. All isolates were tested for susceptibility to eight different antibiotics by the modified Kirby Baeur disc diffusion. Screening and confirmatory tests for ESBL production were done according to Clinical Laboratory Standard Institute (CLSI) guidelines. A total of 25 isolates were Gram-negative which accounted for 41% prevalence, while 59% accounted for Gram-positive. E. coli was the most common isolates (44%), followed by Proteus mirabilis (20%), Klebsiella spp (16%), Enterobacter spp (12%), and Salmonella spp (8%). All isolates displayed multi-drug resistance. The overall production of ESBL-producing isolates was 16%. Our study confirms the prevalence of ESBL-producing uropathogens in Sokoto.
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Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro-iron of hemoglobin (Hb) to ferri-iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi-like approach and the experts panel reached a final consensus >75% of agreement for all the questions. This article is protected by copyright. All rights reserved.
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Background and Objectives: Saudi Arabia has a high prevalence of hereditary hemoglobin disorders. Data has been collected by the Saudi Premarital Screening and Genetic Counseling Program on the prevalence of sickle cell disease and β-thalassemia but the outcomes were not quantified. We used six years of premarital screening data to estimate the burden of sickle disease and β-thalassemia over the program period and to assess the frequency of at-risk marriage detection and prevention. Design and Setting: Retrospective review, premarital couples attending premarital and genetic counseling clinics with marriage proposals between 2004 and 2009. Methods: Blood samples obtained from all couples with marriage proposals between 2004 and 2009 were tested for sickle cell disease and β-thalassemia. Test results were shared with all examinees and genetic counseling was offered for all at-risk couples. Marriage certificates were issued irrespective of the results and compliance with medical advice was voluntary. Results: Out of all men and women examined, 70 962 (4.5%) and 29 006 (1.8%) were carriers or cases of sickle cell disease and β-thalassemia, respectively. While the prevalence of sickle cell disease was constant between 2004 and 2009 (average 45.1 per 1000 examined persons, P=.803), the prevalence of β-thalassemia steadily decreased from 32.9 to 9.0 per 1000 examined persons (P<.001). The frequency of at-risk couples decreased by about 60% between 2004 and 2009 (from 10.1 to 4.0 per 1000 examined persons, P<.001). The frequency of voluntary cancellation of marriage proposals among at-risk couples showed more than 5-fold increase between 2004 and 2009 (from 9.2% to 51.9%, P<.001). The eastern region had 58% of all detected at-risk marriages and showed the greatest decline in detection and increase in prevention over time compared to other regions of Saudi Arabia. Conclusion: Six years of premarital screening in Saudi Arabia markedly reduced the number of at-risk marriages, which may considerably reduce the genetic disease burden in Saudi Arabia in the next decades.
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Introduction: Haemoglobinopathy is a worldwide inherited problem as recognized by WHO and care of affected patients incurs heavy expense on the limited resources of developing countries. There is a need for prevention of births of children with clinically significant haemoglobinopathies by population screening. Cation Exchange-High Performance Liquid Chromatography (CE-HPLC) has emerged to be a simple and precise method to quantify HbA2 , HbF and other variant haemoglobins with certain limitations. Most of the variant haemoglobins can be identified by their retention times, percentages and peak characteristics. Aim: The present study was undertaken to assess the prevalence and spectrum of various haemoglobinopathies in patients reporting to a tertiary health care centre in Uttarakhand, India. Materials and Methods: This was a prospective study conducted on 8144 samples. RBC indices were obtained by sysmex XP 100. CE-HPLC was performed on Biorad D10. The variant haemoglobins were identified on the basis of their percentages, retention times and peak characteristics. Peripheral blood film, reticulocyte count, HbH inclusion and sickling test were done in selected cases. Continuous variables were expressed as mean±SD. Categorical variables were expressed as percentages. Results: Antenatal population formed the bulk of the 8144 cases enrolled in this study. Haemoglobinopathy was seen in 5.9% of the cases with β thalassaemia trait being the commonest abnormality (2.82% of cases). HbD (Punjab) trait was the commonest variant haemoglobin encountered in the study population. There was a significant difference in percentages of variant fractions between compound heterozygotes and variant traits. A presumptive diagnosis of alpha thalassaemia trait was rendered based on RBC indices, iron profile and chromatogram study. Molecular studies were recommended in 81 cases with borderline increase in HbA2 levels to rule out silent mutations. Conclusion: A reasonably high frequency (5.9%) of haemoglobinopathies warrants a routine antenatal screening of total population. An accurate diagnosis can be made in majority of cases by haematological parameters, CE-HPLC chromatograms, cascade screening for haemoglobinopathies and spouses of antenatal cases positive for haemoglobinopathy.
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Introduction: Cerebral vasculopathy, elevated transcranial Doppler velocities and stroke are linked to excessive intravascular haemolysis in sickle cell anaemia. This study determined the prevalence and pattern of abnormal blood flow velocities in children with sickle cell anaemia from Northern Nigeria using transcranial Doppler and to correlate transcranial Doppler velocities with haematological and biochemical markers of haemolysis. Methods: Full blood count, reticulocyte count, fetal haemoglobin and some selected biochemical markers of haemolysis of 100 children with sickle cell anaemia were determined at steady state. The time-averaged mean of maximal velocities in four major intracranial blood vessels was measured using non-imaging transcranial Doppler, which were then classified according to the stroke prevention in sickle cell disease trial protocol. A simple linear correlation between time-averaged mean of maximal velocities in the four major intracranial vessels as the dependent variable and haematological and biochemical markers of haemolysis as independent variables was also determined at the significance level of 0.05. Results: Abnormal transcranial Doppler velocities, classified as high risk, were found in 3% of the studied patients; 11% had intermediate risk while 84% had standard risk. Most high velocities were detected in the middle cerebral artery. The time-averaged mean of maximal velocities of middle cerebral artery positively correlated with total white blood cell count, absolute neutrophil count, platelet count, reticulocyte count, serum lactate dehydrogenase and total bilirubin, while it was negatively correlated with haematocrit and fetal haemoglobin levels. Conclusion: Our study showed low prevalence of abnormal transcranial Doppler velocities and low prevalence is unrelated to low markers of haemolysis in our study population.
Article
Introduction The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15 years. Materials and Methods Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. Results The study includes 31 075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14 423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ)⁰ Indian inversion and the HPFH‐3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (−α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified. Conclusion The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.
Chapter
The electrophoresis on cellulose acetate membrane is most widely used because of its simplicity, and is without the use of any sophisticated instrument other than electrophoresis apparatus and the cellulose acetate strip. Here we describe a modified version of cellulose acetate membrane electrophoresis for hemoglobin separation from blood sample. Sharp, clear bands without tailing effects can be obtained with this method. The method and apparatus described here would be appropriate to separate protein fractions under 1 h at voltages up to 60 V/cm measured between the electrodes.
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Hb F levels were determined on samples from 750 normal blood donors. Six individuals (0.8%) had Hb F levels in excess of 1.1%, the upper end of the continuous distribution. Eight individuals at the upper end and 7 individuals at the lower end of the range were selected for family studies. These studies revealed that the control of Hb F levels in adults, as judged by the more sensitive F-cell technique, has a major genetic component. Structural analysis of the Hb F in several cases demonstrated that both G gamma and A gamma chains were present, but in variable proportions. These results are discussed in light of current concepts of adult F-cell production.
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Specific antibodies to human fetal hemoglobin were prepared and, after conjugation with a fluorescent dye, were used to determine the distribution of Hb F-containing cells in blood smears from normal adults and individuals with hereditary and acquired conditions associated with abnormal levels of Hb F. The mean proportion of F-cells in normal persons was 2.7% +/- 1.4%, with a range of 0.5%-7.0%. Proportions of F-cells were increased in persons with several acquired and inherited disorders that are associated with an increased percentage of Hb F in hemolysates. A strong linear correlation between the amount of Hb F and proportion of F-cells was observed. This technique may prove useful in studies of a variety of disorders associated with Hb F elevations and also in investigations of the mechanisms controlling the transition from fetal to adult hemoglobin during the course of human development.
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An enzyme-linked immunosorbent assay (ELISA) for the measurement of nanogram quantities of hemoglobin A and hemoglobin F is described. The assay employs hemoglobin bound to polystyrene test tubes. The antigen is quantitated by the addition of rabbit antibody specific for the hemoglobin type, followed by a second antibody of goat antirabbit gamma globulin coupled to alkaline phosphatase. Quantitation of the reaction is achieved by incubating the coated tubes with p-nitrophenyl phosphate. The binding characteristics, the linearity, the ability of the assay to discriminate between hemoglobin A and hemoglobin F, the ability to discriminate between native and denatured gamma globin, and the comparison of this assay with a previously described immunoradiometric (IRMA) assay are discussed.
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A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in approximately 90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the "beta Thalassemia Short" program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.
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Iron-deficiency anemia and thalassemia are among the most common microcytic anemias. Differentiating these anemias by means of hemogrant indices is imprecise. Powerful statistical computer programming now enables sensitive discriminant analyses to aid in the diagnosis. Laboratory results from 383 adults were examined retrospectively and grouped according to their original diagnoses: normal (n = 78); beta-thalassemia (n = 134); alpha-thalassemia (n = 106); and iron-deficiency anemia (n = 65). Statistical analysis of results evaluated only RBC indices: RBC count, hemoglobin level, mean corpuscular volume, mean corpuscular hemoglobin, and RBC distribution width. Stepwise multivariate discriminant analysis determined those indices that best differentiated the 4 groups. The Fisher linear discriminant function for each group was calculated and tested casewise. Discriminant analysis identified mean corpuscular hemoglobin, RBC count, mean corpuscular volume, and RBC distribution width as the best set of indices for differentiating the 4 diagnoses. Casewise testing of the calculated Fisher linear discriminant function resulted in mean-weighted sensitivity of 80.4%. The present study demonstrates that a set of linear discriminant functions based on routine hemogram data can effectively differentiate between alpha-thalassemia, beta-thalassemia, and iron-deficiency anemia, with a high degree of accuracy.
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To investigate the costs and potential benefits of different policies for antenatal screening for haemoglobinopathies in two multiethnic London communities. 1000 consecutive antenatal patient samples referred to each of two London teaching hospital laboratories for haemoglobinopathy testing were investigated using the standard procedures of the laboratory in question. When the standard procedures did not include high performance liquid chromatography (HPLC), this technique was added, in order to assess its diagnostic value and cost-effectiveness. A comparison was made between the costs and potential benefits of universal testing for variant haemoglobins and beta thalassaemia trait using HPLC and the costs and potential benefits of universal testing for variant haemoglobins and selective testing for beta thalassaemia trait using the mean cell haemoglobin (MCH) as a screening test and less automated techniques than HPLC for definitive diagnosis. The costs of the two policies were found to be comparable, as the higher reagent/instrument costs of HPLC were offset by the lower labour costs. Universal testing of 2000 consecutive samples did not disclose any extra cases of beta thalassaemia trait which would not have been detected by universal screening and selective testing. However, six patients were found to have a haemoglobin A2 variant which can interfere with the diagnosis of beta thalassaemia trait. The introduction of universal testing by HPLC into British laboratories could be cost neutral and has potential benefits. If a higher cost is accepted then the greater degree of automation could be used to release skilled staff for other tasks within the laboratory.
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Universal antenatal haemoglobinopathy screening in this hospital has identified several women with increased haemoglobin A(2) values, but without hypochromic microcytic red cell indices. This report describes two cases where there is evidence that the raised haemoglobin A(2) value is not caused by heterozygous beta thalassaemia, but rather results from these patients being human immunodeficiency virus (HIV) positive and on antiretroviral therapy. This will have important implications as universal antenatal haemoglobinopathy screening becomes more widespread, and as the number of HIV positive women of childbearing age increases.
Article
The laboratory diagnosis of haemoglobinopathies, including the thalassaemias, is of growing importance, particularly because of an increasing requirement for antenatal diagnosis of significant disorders of globin chain synthesis. This guideline discusses the laboratory tests which are most useful in the diagnosis of haemoglobinopathies and describes their role in specific clinical circumstances. Of the newer technical methods, high-performance liquid chromatography (HPLC) is of considerable importance whereas isoelectric focusing (IEF) and immunoassay for variant haemoglobins have a more minor role. Specific recommendations have been formulated for testing in relation to genetic counselling and for neonatal diagnosis. Methods used in specialized laboratories for fetal diagnosis have been tabulated. Genetic counselling requires: (i) identification of haemoglobins S, C, D-Punjab, O-Arab, E, Lepore and H, and (ii) the detection of carriers of α° and β thalassaemia. It is recommended that subjects of all ethnic groups be screened for β-thalassaemia trait, all except Northern European Caucasians for variant haemoglobins, and selected ethnic groups for α°-thalassaemia trait. Testing for β-thalassaemia trait should be carried out when the mean cellular haemoglobin (MCH) is < 27 pg and testing for α°-thalassaemia trait should be considered when the MCH is < 25 pg. Appropriate methods include HPLC or haemoglobin electrophoresis for identification of variant haemoglobins and HPLC or microcolumn chromatography for quantification of haemoglobin A2.
A complete spectrum of genetic lesions affecting the β-globin gene giving rise to a complete spectrum of phenotypic severity is described. Although most of the molecular lesions involve the structural β gene directly, some down regulate the gene through in-cis effects at a distance while trans-acting factors are implicated in a few cases. The remarkable phenotypic diversity can be related ultimately to the degree of α-globin-β-globin chain imbalance and arises from variability of mutations affecting the β gene itself and from interactions with other genetic loci, such as the α- and γ-globin genes. The presence of other interacting loci is implicated by their interactions in increasing γ gene expression or by an increased proteolytic capacity of the erythroid precursors. It is hoped that observations from the genotype-phenotype relationship might form the basis for a comprehensive diagnostic database that will be useful not only for genetic counselling and prenatal diagnosis but also for providing prognostic information for decision making in bone marrow transplantation and gene therapy programmes in the future. However, it is clear from recent analyses that, apart from the two categories of triplicated α genes with heterozygous β-thalassaemia and inheritance of mild β+-thalassaemia alleles, it is still not possible to predict consistently phenotype from α and β genotypes alone owing to the influence of the other modulating factors, some implicated (such as inheritance of hereditary persistence of fetal haemoglobin) and others as yet unidentified.
The large number of naturally occurring mutants of this well-characterized locus provides an excellent opportunity for elucidating the relationship between its structure and function. Comparisons of what has been learned about the α-globin locus with complementary observations on the β-globin locus, provide a strategy for understanding the co-ordinate regulation of eukaryotic gene expression. From a practical point of view it is important to remember that millions of individuals throughout the world are carriers of α-thalassaemia and every year many thousands of pregnancies are at risk of producing children with the severe α-thalassaemia syndromes. The data summarized here provide the basis for accurately predicting the genotype in such cases and thus enabling appropriate prenatal testing. However, because this is a genetic disease that predominantly affects individuals from countries with limited health resources, simpler and cheaper methods of screening and diagnosis will have to be developed before this information has a significant impact on the attendant morbidity and mortality (see Chapter 9, this volume).
Article
Radial immunodiffusion techniques for hemoglobins F and A2 are described. Both techniques compare favorably with results obtained by alkali denaturation and cellulose acetate electrophoresis, respectively. Comparable results are obtained by immunoassay of hemoglobin solutions or lysed whole blood, obviating expensive equipment or washing red cells.
Article
EXAMINATION of the blood of a ten-year-old girl with congenital Heinz-body anæmia has provided evidence for the presence in the subject's red cells of an unstable hæmoglobin fraction.
Article
Using a method involving elution of hemoglobin bands from cellulose acetate strips following electrophoresis of hemolysates, hemoglobin A2 (Ab A2) was quantitated in bloods from 300 healthy individuals and 904 patients. The percentage of Hb A2 was elevated in beta-thalassemia heterozygotes and some patients who had megaloblastic anemia. In the latter, the highest Hb A2 levels were observed in patients with the most severe anemia. Low Hb A2 percentages were found in iron-deficiency anemia, hereditary persistance of fetal hemoglobin, and Hb H disease. In iron-deficiency anemia, the lowest levels of Hb A2 were observed in association with the most severe anemia. Iron and folate deficiency each suppressed Hb A2 levels in beta-thalassemia heterozygotes; however, vitamin B12 deficiency did not alter the percentage of Hb A2 in thalassemia. Malignant tumors, renal and hepatic insufficiency, chronic infections and inflammation, hemolytic disease, lead poisoning, aplastic anemia, leukemia, myelofibrosis, and hypothyroidism did not change Hb A2 levels. The pathogenesis of altered Hb A2 levels and their clinical significance in various diseases are discussed.
Article
Erythrocytosis resulting from the presence of a haemoglobin molecule having an increased affinity for oxygen is uncommon but it may be diagnosed more frequently when suitable screening tests are used more widely. The best screening procedure is the measurement of the P50 which only requires 3 ml of venous blood, but if this test is not available, haemoglobin electrophoresis at both alkaline pH (cellulose acetate) and acid pH (agar) will detect some of these high affinity variants. The possibility of a high affinity haemoglobin should be considered in any patient with polycythaemia without splenomegaly or thrombocytosis and in whom there is no underlying disease known to cause polycythaemia. Correct diagnosis should lead to a rational approach to the management of these patients.
Article
Isoelectric focusing on slabs of acrylamide gel was adapted for the screening of abnormal hemoglobins, the characterization of 70 human variants, and the study of minor fractions of normal hemoglobin. The screening method was as fast and inexpensive as conventional techniques, allowed the simultaneous analysis of some 50 samples of whole blood, and yielded resolution superior to that obtained by other methods with hemolysates. Among the 70 variants, 31 mutants could not be separated from HbS by cellulose acetate electrophoresis. The characterization technique of electrofocusing allowed us to distinguish between most variants. Only one mutant, Hb Galveston, could be confused with HbS. Hb Köln, the most frequent unstable mutant, exhibited a special pattern. HbA1C was separated from HbA. Preliminary results indicate that quantitation of HbA1C by gel scanning is feasible.
Article
A modification of the microchromatographic procedure for Hb-A2 which utilizes DEAE-cellulose (Reference 1) allows the quantitation of Hb-A2 without interference from any Hb-S in the sample. Elution of Hb-A2 with a glycine-KCN developer is much less sensitive to minor change in pH of the developer, but is greatly dependent on the pH of the ion exchanger.
Article
Electrophoresis of the globin chains of hemoglobin on cellulose acetate in both acidic and alkaline buffers (pH about 6 and 9) containing urea and 2 mercaptoethanol is a simple, rapid means of characterizing hemoglobins. Erythrocyte hemolysate is electrophoresed in the presence of a large amount of mercaptoethanol, which liberates heme from globin, and keeps it in solution during its rapid electrophoretic removal. Each globin chain migrates at a characteristic rate, which varies with the pH and composition of the buffer. The combined data permit differentiation, with a high degree of specificity, of some similarly charged hemoglobins. They may also be useful in assessing the effect of secondary and tertiary structure on molecular charge.
Article
. A simple test is described for the detection of unstable haemoglobins by the addition of haemolysate to an isopropanol/buffer solution at 37°C. Over 200 normal haemolysates have been tested giving no precipitation up to 30 min incubation whilst six samples containing an unstable haemoglobin gave a precipitate within 5 min, which was flocculent by 20 min. As well as being a simple and effective screening test, the procedure is suitable for the purification of unstable haemoglobins for further studies but suffers from limitations, probably in common with the heat precipitation method, in the quantitation of the abnormal haemoglobin. Its use as a research tool is illustrated by comparative measurements of the stabilities of various forms of haemoglobin. These show deoxyhaemoglobin to be considerably more stable than oxyhaemoglobin thus supporting suggested interchain bondings in deoxyhaemoglobin.
Article
By means of a simple microchromatographic method, Hb-A2 may be rapidly and accurately determined in blood samples from subjects with and without sickle cell hemoglobin. The procedure makes use of DEAE-cellulose, inexpensive laboratory glassware, and allows one technician to determine the level of Hb-A2 in at least 50 samples a day. Whole blood samples, red cell hemolysates, and blood collected on filter paper can be used without dialysis and can be analyzed with comparable accuracy.
Article
I DENTIFICATION of the abnormal polypeptide chain of a hemoglobin ( Hb ) variant is often useful in connection with structural analyses and Chernoff and Pettit' have recently improved on existing methods for this purpose by using starch gel electrophoresis of globin in urea barbital buffer. Izzo2 used this same buffer for electrophoresis of globin on cellulose acetate. Ohba et al.3 have proposed starch gel electrophoresis of whole hemolysates in the presence of p-chloromercuribenzoic acid. We now report a simple, rapid method of differentiating polypeptide chains of hemoglobin by cellulose acetate electrophoresis of whole hemolysates in urea barbital buffer.
Article
A rapid and reproducible electrophoretic method for the separation and quantitation of haemoglobins on cellulose acetate is described. The accuracy of the method and its possible sources of error are discussed. The normal range for haemoglobin A(2) by this method is 1% to 3% of the total haemoglobin concentration. Blood samples from 32 thalassaemic patients showed haemoglobin A(2) values of 3.5% to 7%.
Considerable advances have been made recently in our understanding of globin gene expression, its developmental regulation and the altered patterns in various inherited and acquired disorders. Each advance has revealed a new layer of complexity and as many questions remain as have been answered. Adult levels of HbF are clearly under genetic control but the number and nature of these genetic factors, either within or outside of the β-globin gene cluster, remain to be determined. Many of the conditions resulting in increased HbF in adult life appear to involve an increased erythropoietic drive which results in a higher proportion of erythroid progenitor cells activating their inherent ability to synthesise low amounts of HbF. The mechanism by which this is achieved remains unknown but these observations have been confirmed in a number of experimental systems and have led to the use of mildly cytotoxic drugs to increase the HbF levels in sickle cell anaemia. Similarly, the clinical observation that infants of diabetic mothers show delayed fetal to adult haemoglobin switching has led to the development of butyrate derivatives to increase adult HbF levels.
Article
We observed increased hemoglobin A2 (HbA2) levels in an asymptomatic human immunodeficiency virus-1 (HIV1) patient with no previous history of beta-thalassemia. He was treated only with zidovudine (AZT). In an attempt to understand this observation, a retrospective study was initiated to determine whether mean HbA2 levels are higher in AZT-treated patients than in subjects not receiving this drug and to assess if other hematologic alterations are associated with elevated HbA2. One hundred fifty-one HIV-positive cases were investigated; AZT was administered to 81 of them. The mean value of HbA2 was 0.032 (SD +/- 0.005) for the treated group vs. 0.027 (SD +/- 0.004) for the controls. This difference was highly significant (P < 0.001). Twenty-four patients (31%) in the treated group had elevated HbA2 levels vs. none in the controls. Bone marrow toxicity seemed to be more significant in patients with heightened HbA2 values, and HbA2 levels did not increase with CDC clinical stage. We conclude that AZT may be linked to high HbA2 levels in some patients.
Article
The differentiation between thalassemic and non-thalassemic microcytosis has important clinical implications in hematology and medicine. A simplified index, based on red cell parameters derived from automated blood cell analyzers, which could be used to discriminate between microcytic patients with a high probability of thalassemia minor and those with a low probability, would be an extremely useful tool. Five mathematical indices have been proposed as useful for this purpose. These are the: Bessman index, Shine and Lal index, England index, Mentzler index, and mean cell volume (MCV) alone. This study was designed to prospectively evaluate the efficacy of these indices. Patient samples were chosen every fourth day from all patient samples referred to the hematology laboratory at St. Joseph's Hospital over a 6-month period. All patient samples with an MCV < 80 fL and age > or = 18 years were considered eligible for the study. After enrollment and laboratory analysis were complete sensitivities and specificities were calculated for each of the indices using a variety of cut-off values and receiver operator characteristic (ROC) curves were constructed. Based on statistical analysis of the area under these curves, the authors conclude that MCV alone is as effective as the Mentzler and Shine and Lal indices in selecting microcytic patient samples with a high probability of thalassemia minor for thalassemia testing. They also conclude that the Bessman index and the England index are ineffective indices for this purpose.
Article
The introduction of automation for haemoglobinopathy screening is an important advance in technology for haematology laboratories. This paper evaluates the utility of an automated HPLC instrument, the Bio-Rad 'Variant' for the detection and quantitation of the normal haemoglobins (Hb A, A2 and F) and the common abnormal haemoglobins (Hb S, C, DPunjab, E, OArab and Lepore) which need to be evaluated in laboratories undertaking carrier and/or neonatal screening for sickle cell and thalassaemia. The instrument only uses a small amount of whole blood (5 microliters), a 3 mm disc from a Guthrie spot may also be used for analysis of samples from neonates. It uses a 100 place automatic sampler with a cycle time of 6.5 min for adult samples (using the 'Beta Thalassaemia Short' reagent pack) and 3 min for neonatal samples. The automatic sampler also allows samples to be analysed 'out of hours'. A 'STAT'; position allows urgent samples to be analysed before, or during, a routine analytical run. All reagents, other consumables and application notes are provided by the suppliers. Other types of reagent packs, such as the 'Sickle Cell Short' for neonatal screening were not assessed during this study.
Article
The precise identification of human hemoglobin variants, over 700 human hemoglobin variants are known, is essential for prediction of their clinical and genetic significance. A systematic approach to their rapid identification is described. Traditionally this requires protein or DNA characterization which entails lengthy analytical procedures. To overcome these obstacles a rapid approach to variant hemoglobin identification has been developed using conventional phenotypic methods combined with electrospray ionization-mass spectrometry (ESI-MS). The latter requires only a small amount of whole blood (10 microl) but in most cases 2 microl would have been sufficient and no preanalytical steps, such as separation of red cells or globin chains, are necessary. Aged, hemolyzed blood samples can also be analyzed. This approach has been used to positively identify 95% of the variants in over 250 samples. The remaining 5% in which a variant was detected by phenotypic techniques were not resolved by mass spectrometry. Ninety-nine different abnormalities comprising 36 alpha-chain variants, 59 beta-chain variants (including 2 extensions), and 4 hybrid hemoglobins were identified. These include 15 novel variants. The application of ESI-MS described requires approximately 1 h to prepare and analyze each sample and has minimal reagent costs. The turnaround time on a single sample can be as little as 2 h. This technique can now be considered a useful additional tool for reference laboratories.
Article
A general practitioner requested a full blood count and haemoglobin electrophoresis on a 38-year-old Somalian woman with Ôknown anaemiaÕ. The haemoglobin concentration was normal, although there was borderline microcytosis. High performance liquid chromatography (HPLC) was performed as per standard protocol in our laboratory. The trace (lower image) showed the presence of haemoglobins A, A 2 and F quantified at 80%, 2AE4% and 2AE4% respectively. In addition, there was a prominent peak of glycosylated haemoglobin (arrowhead, compared with a normal trace, upper image), quantified at 10AE9%. The patient's general practitioner was contacted. The patient was not known to be diabetic but she did have a history of dizziness, weight loss, thirst and urinary frequency. Appropriate samples were subsequently obtained and these showed a fasting blood glucose of 16AE9 mmol ⁄ l and HbA 1c of 11AE4%, confirming the diagnosis of diabetes mellitus. This case illustrates that using HPLC rather than haemoglobin electrophoresis as the primary technique for investigating suspected haemoglobinopathies may lead to the diagnosis of unsuspected diabetes mellitus. Haemoglobinopathy laboratories should therefore draw the attention of clinical staff to an increase of glycosylated haemoglobin in any patient who is not known to be diabetic.
Article
IN the alkali denaturation method of Singer, Chernoff and Singer1 the one-minute residue of normal adult hæmoglobin gives a spectrophotometric reading of 0.5-1.8 per cent `alkali resistant hæmoglobin'. It is well known1,2,3, that most of this residue is not true foetal hæmoglobin, but some other hæm-compound, the nature of which is unknown.
Article
Large, usually single inclusions, having the staining properties of Heinz-bodies and occurring spontaneously in thalassemic cells, are described. Their frequency is greatest in the normoblasts, either circulating ones or those found in the bone marrow, and decreases sharply in more mature red cells. Splenectomy unmasks this inclusion phenomenon to a large extent. Some staining and other properties of these inclusion bodies are described. It is concluded that these inclusions represent precipitated hemoglobin, very likely uncombined α-chains. The findings are discussed and related to present-day knowledge on hemoglobin synthesis and erythrocyte turnover in thalassemia.
Article
As well as six 'normal' haemoglobins that occur at various stages of development, more than 800 abnormal or variant haemoglobins have been described. Many of these variant haemoglobins have no significant clinical consequences apart from causing confusion to clinicians and in laboratories; however, some of the variant haemoglobins result in major morbidity or mortality. The laboratory challenge is to detect these clinically significant haemoglobins and to identify them with sufficient accuracy for clinical purposes, as well as to quantitate both these and the 'normal' haemoglobins. The techniques used to detect and quantitate these haemoglobins in routine service laboratories are discussed in detail. Methods used by referral laboratories, such as mass spectrometry and DNA analysis, are briefly discussed. Haemoglobin analysis is most often undertaken as part of neonatal, antenatal or pre-anaesthetic screening; these programmes are reviewed, together with possible changes to neonatal screening and antenatal screening that may occur as part of the NHS National Plan.